Protein-protein interactions (PPIs) are essential to vital cellular processes, and are involved in numerous patophysiological states, where they serve as potential targets for therapeutic intervention. PPIs have generally been perceived as difficult to target with therapeutic molecules, since they are often characterized by large, flat, and hydrophobic interfaces.
A class of PPIs is one involving PDZ domains [PDZ is an abbreviation for postsynaptic density protein-95 (PSD-95), Drosophila homologue discs large tumor suppressor (DlgA) and zonula occludens-1 protein (ZO-1)]. PDZ domains often function as modules in scaffolding proteins that are involved in assembling large protein complexes in the cell, and are highly abundant in eukaryotic organisms. PDZ domains comprise about 90 amino acids and generally interact with the C-terminal of the interacting protein. PSD-95, contains three PDZ domains, PDZ1-3, which bind peptide ligands with the consensus sequence Glu/Gln-Ser/Thr-X-Val-COOH.
The structural basis for the interaction of PDZ domains with C-terminal peptides was first elucidated by an X-ray crystallographic structure of PDZ3 of PSD-95 complexed with a native peptide ligand, CRIPT (Sequence: YKQTSV (SEQ ID NO: 3)). PDZ3 contains six antiparallel β-strands (βA-βF) and two α-helices (αA and αB), and the C-terminal peptide ligand binds as an additional anti-parallel β-strand into a groove between the βB strand and αB helix. Two residues in the peptide ligand are considered particularly important for affinity and specificity, the first (P0) and the third (P−2) amino acids (counting from the C-terminal). The side chain of the amino acid in P0 position projects into a hydrophobic pocket and an amino acid with an aliphatic side chains (Val, Ile and Leu) is required. In the PDZ3-CRIPT structure, the hydroxyloxygen of Thr (P−2) forms a hydrogen bond with the nitrogen of an imidazole side chain of His372. A conserved Gly-Leu-Gly-Phe (SEQ ID NO: 15) (position 322-325 in PDZ3) motif and a positively charged residue (Arg318 in PDZ3) of PDZ domains mediate binding to the C-terminal carboxylate group.
The PDZ1 and PDZ2 domains of PSD-95 interact with several proteins including the simultaneous binding of the N-methyl-D-aspartate (NMDA)-type of ionotropic glutamate receptors and the nitric oxide producing enzyme, neuronal nitric oxide synthase (nNOS) (FIG. 1). NMDA receptors are the principal mediators of excitotoxicity, which is implicated in neurodegenerative diseases and acute brain injuries, and although antagonists of the NMDA receptor efficiently reduce excitotoxicity by preventing glutamate-mediated ion-flux, they also prevent physiological important processes. Thus NMDA receptor antagonists have failed in clinical trials for stroke due to low tolerance and lack of efficacy. Instead, specific inhibition of excitotoxicity can be obtained by perturbing the intracellular nNOS/PSD-95/NMDA receptor complex with PSD-95 inhibitors (FIG. 1). PSD-95 simultaneously binds the NMDA receptor, primarily GluN2A and GluN2B subunits, and nNOS via PDZ1 and PDZ2. Activation of the NMDA receptor causes influx of calcium ions, which activates nNOS thereby leading to nitric oxide (NO) generation. Thus, PSD-95 mediates a specific association between NMDA receptor activation and NO production, which can be detrimental for the cells if sustained for a longer period, and is a key facilitator of glutamate-mediated neurotoxicity (FIG. 1). Inhibition of the ternary complex of nNOS/PSD-95/NMDA receptor interaction by targeting PSD-95 is known to prevent ischemic brain damage in mice, by impairing the functional link between calcium ion entry and NO production, while the physiological function, such as ion-flux and pro-survival signaling pathways, of the NMDA receptor remains intact.
Inhibition of the nNOS/PSD-95/NMDA receptor complex has previously been achieved with a nonapeptide, corresponding to the C-terminal of GluN2B, fused to HIV-1 Tat peptide, known for its ability to facilitate membrane and blood-brain barrier permeability. This 20-mer peptide (Tat-NR2B9c; Sequence: YGRKKRRQRRRKLSSIESDV (SEQ ID NO: 4)) has shown promising neuroprotective properties in rat models of ischemic brain damage (Aarts et al., Science 298, 2002, p. 846-850, 2002; Sun et al., Stroke 39, 2008, p. 2544-2553) and is currently in clinical trials as a potential drug for the treatment of cerebrovascular ischemia, as seen in stroke. However, this compound suffers from low affinity (Ki=4.6 μM; see later) to PDZ1-2 of PSD-95, which potentially makes it an inefficient and non-selective compound.
WO2010/004003 describes dimeric peptide ligands linked by a polyethylene glycol linker (PEG) that simultaneously bind to the PDZ1 and PDZ2 domains of PSD-95 and their use for treatment of cerebrovascular ischemia. There remains a need for PSD-95 inhibitors with a higher affinity for PDZ1 and PDZ2 domains, and that have an improved therapeutic effect in vivo for the treatment of treatment of ischemic stroke and traumatic brain injury.
Neuropathic pain is caused by damage to the peripheral or central nervous system due to traumatic injury, surgery, or diseases such as diabetes or autoimmune disorders. Such damage leads to an acute phase response characterized by ‘nociceptive pain’ and inflammation. In a large proportion of patients, pain persists despite injury healing, resulting in a state of chronic neuropathic pain. In addition to the involvement of inflammation after nerve injury, chronic pain may also be initiated by inflammation induced by mediators released by immune cells, which cause a sensitization of pain pathways. Sensitization of spinal sensory neurons (‘wind-up’) is a shared feature of neuropathic pain and chronic inflammatory pain, and is evoked by a prolonged activation of nociceptors. The symptoms present as spontaneous burning pain, an exaggerated response to painful stimuli (hyperalgesia), and pain in response to normally non-painful stimuli (allodynia). Chronic pain, particularly as a result of nerve injury, is poorly managed by current drugs such as opioids and non-steroidal anti-inflammatory drugs (NSAIDs). NMDA receptor antagonists block sensitization of pain responses and display good analgesic properties in animal models and clinical settings, but they are associated with unacceptable side-effects and can therefore not be used clinically. Accordingly there is a need for alternative drugs capable of providing improved pain treatments, particularly NMDA receptor related pain symptoms, while avoiding the unacceptable side-effects of current drugs.